JP2006109735A - Wheat gluten-accumulating transformed rice plant and method for creating the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a wheat substitute plant which accumulates proteins participating in bread productivity. <P>SOLUTION: This transformed rice plant containing the following gene (a) or (b) encoding wheat glutenin protein GluD1 y type 10. (a) The gene comprising a DNA having a specific base sequence, or (b) the gene encoding a protein comprising a specific amino acid sequence. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to the production of cultivar-improved rice that accumulates GluD1 y type No. 10, one of the glutenin subunits that are wheat-derived proteins involved in bread-making properties.

  Wheat flour produced from wheat kernels is the main ingredient of bread. Proteins contained in wheat flour include glutenin, gliadin, and albumin. Of these, glutenin is considered to be the most important ingredient for producing high-quality bread. Glutenin is a polymer composed of various low molecular and high molecular weight glutenin subunits, and has high elasticity. Various types of base sequences have already been determined and reported for genes encoding the proteins contained in wheat flour (for example, Non-Patent Document 1).

  In recent years, bread has become popular in Japan, but the self-sufficiency rate of wheat, which is a raw material for bread, has remained very low. This is partly because Japan's climate is not well suited for wheat cultivation. In Japan, wheat is planted in the fall, except in parts of Hokkaido, and begins to grow around mid-March in early spring. However, since the rainy season already begins in June, there is only a net three months for growing wheat, opening flowers, and fruit-filling, making it difficult to increase wheat yield in Japan. In order to stably produce high-quality wheat flour in Japan, the production of new wheat varieties that have high bread-making characteristics and high yields can be expected even in the Japanese climate, or high bread-making characteristics and high yields in the Japanese climate. Production of wheat substitute plants is desired.

Traditionally, the production of new plant varieties has been based on cross breeding or mutation breeding. However, breeding by mating and mutation basically requires individual differences and chances, so the success rate of breeding is not necessarily high, and a great deal of cost and time are required to obtain the desired breed. It was necessary. In recent years, new plant varieties have been produced using genetic engineering techniques to solve this problem. Furthermore, with the development of genetic engineering techniques, it has become possible to provide functions between varieties that cannot be crossed. For example, as an attempt to express and accumulate foreign gene products in rice, there have been many reports such as studies on the expression of soybean glycinin protein (Patent Document 1). However, no new wheat varieties or wheat substitute plants that have achieved high bread-making properties and high yield have been reported so far.
JP 2000-50871 A Anderson OD et al., Nucleic Acids Research (1989) 17 (1), p.461-462

  An object of the present invention is to provide a wheat substitute plant that accumulates proteins involved in bread-making properties.

  Paddy field cultivation has many features not found in field cropping. In field cropping, there is a high possibility of causing a continuous cropping disorder in which specific components are deficient, waste products are accumulated, and organisms causing disease are increased. On the other hand, in paddy fields, the missing components are brought in by irrigation water, and waste products are washed away. Disease-causing organisms are difficult to survive in water-covered soil. Paddy fields also help to mitigate flooding by mitigating temperature fluctuations. Conversely, in upland fields, the topsoil is carried away by rainwater and wind, causing soil erosion and loss.

  In addition, in Japan, when rice is grown using full paddy fields, although 15 million tons of rice can be produced per year, the recent decline in rice consumption has led to approximately 40% (6 million) of paddy fields. Tons) is subject to rice planting restrictions. On the other hand, wheat production is extremely low per unit area in Japan, with imports reaching 6 million tons per year. This amount of import is equivalent to the area that is subject to rice planting restrictions.

  Furthermore, in Japan, rice is weaker than wheat and transplanted to paddy fields at the beginning of May, but it grows in the summer and grows leaves and leaves slowly until mid-September. Thus, rice grows over a net period of 5 months, so it is obvious that the yield is higher than that of wheat, which has a net growth period of about 3 months.

  Therefore, the present inventors can produce flour even under a Japanese climate that is not generally suitable for wheat cultivation, if a high bread-making wheat substitute plant can be produced using rice having various advantages in cultivation. We thought that it would be possible not only to produce substitutes efficiently, but also to make effective use of rice fields that are restricted to rice cultivation, which would be useful in promoting agriculture.

  Based on this idea, the present inventors have made extensive studies to solve the above-mentioned problems. As a result, the GluD1 y type No. 10 glutenin subunit protein (wheat glutenin GluD1) that sits on the D genome of wheat. Select a gene encoding y type No. 10), isolate it from the wheat genome, insert it into a plant transformation vector, introduce it into rice cells using the Agrobacterium method, and transform to select hygromycin-resistant rice Later, when the presence or absence of accumulation of glutenin protein in the rice grain was verified, the present inventors succeeded in obtaining transformed rice that accumulates wheat glutenin protein in the rice grain. Since this transformed rice was shown to produce cereal (bread rice) as a raw material for high-quality wheat flour showing high bread-making properties, the present invention was completed.

That is, the present invention is as follows.
[1] A transgenic rice containing the following gene (a) or (b) encoding wheat glutenin protein GluD1 y type 10:
(a) a gene consisting of DNA having the base sequence represented by SEQ ID NO: 1, or
(b) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2
[2] The transformed rice according to [1] above, wherein the rice cultivar is Nipponbare.
[3] A method for producing highly-baked transformed rice, which comprises introducing the following gene (a) or (b) encoding wheat glutenin protein GluD1 y type 10 into rice cells:
(a) a gene consisting of DNA having the base sequence represented by SEQ ID NO: 1, or
(b) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2
[4] A recombinant vector for imparting high bread-making property to rice, comprising the following gene (a) or (b) encoding wheat glutenin protein GluD1 y type 10:
(a) a gene consisting of DNA having the base sequence represented by SEQ ID NO: 1, or
(b) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2

  The transformed rice according to the present invention can accumulate wheat glutenin protein exhibiting high bread-making property in rice grains. That is, the transformed rice according to the present invention can produce cereal (bread rice) as a raw material for a high-quality wheat flour substitute. And, by using the method for producing high bread-producing transformed rice according to the present invention, a high bread-producing wheat substitute plant that can be cultivated even in land that is not suitable for wheat cultivation can be easily produced. Can do. In such a method, a recombinant vector containing a gene encoding wheat glutenin GluD1 y type No. 10 for imparting high bread-making properties to the rice according to the present invention is very suitably used for gene introduction. be able to.

1. Gene encoding wheat glutenin GluD1 y type 10 In the present invention, a gene encoding a glutenin subunit protein (wheat glutenin GluD1 y type 10) of GluD1 y type 10 is introduced into rice.

  The gene encoding wheat glutenin GluD1 y type 10 according to the present invention is not limited, but typically encodes a glutenin subunit present at the Glu-D1 locus on the wheat D genome. One of the alleles. Preferably, the gene encoding wheat glutenin GluD1 y type 10 according to the present invention is a gene consisting of DNA having the base sequence shown in SEQ ID NO: 1, or the amino acid sequence shown in SEQ ID NO: 2 (648 amino acids) A gene encoding a protein consisting of The base sequence of the gene encoding wheat glutenin GluD1 y type No. 10 shown in SEQ ID NO: 1 has already been reported as accession number X12929 in the international base sequence database (Non-patent Document 1). In the base sequence of SEQ ID NO: 1, base numbers 617 to 619 are start codons, and base numbers 2561 to 2563 are stop codons. The gene encoding wheat glutenin GluD1 y type 10 of the present invention is one or several (for example, 2 to 10) in the amino acid sequence shown in SEQ ID NO: 2 as long as the glutenin physical property of GluD1 y type 10 protein is maintained. It may be a gene encoding a protein consisting of an amino acid sequence in which these amino acids are deleted, substituted or added. In addition, the gene encoding wheat glutenin GluD1 y type No. 10 of the present invention hybridizes under stringent conditions with the DNA having the base sequence shown in SEQ ID NO: 1 and retains the glutenin physical properties of GluD1 y type No. 10. It may be a gene consisting of DNA encoding the protein to be processed.

  In the present invention, “stringent conditions” refers to conditions under which a so-called specific hybrid is formed. For example, nucleic acids having high homology, that is, DNAs having 90% or more, preferably 95% or more homology, hybridize, and nucleic acids having lower homology do not hybridize. More specifically, the sodium salt concentration is 15 to 750 mM, preferably 50 to 750 mM, more preferably 300 to 750 mM, the temperature is 25 to 70 ° C., preferably 50 to 70 ° C., more preferably 55 to 65 ° C., This refers to conditions at a formamide concentration of 0-50%, preferably 20-50%, more preferably 35-45%. Furthermore, under stringent conditions, the washing conditions of the filter after hybridization are usually 15 to 600 mM, preferably 50 to 600 mM, more preferably 300 to 600 mM, and a temperature of 50 to 70 ° C., preferably It is 55-70 degreeC, More preferably, it is 60-65 degreeC.

  In the present invention, “gene” includes DNA and RNA. Further, DNA includes at least genomic DNA and cDNA, and RNA includes mRNA and the like. The “gene” of the present invention may contain a sequence such as a sequence of an untranslated region (UTR) in addition to a sequence encoding wheat glutenin GluD1 y type # 10.

  The gene encoding wheat glutenin GluD1 y type No. 10 according to the present invention is derived from wheat (preferably cultivar Max) exhibiting a high bread-making property using, for example, a primer designed based on the sequence of SEQ ID NO: 1 or 2. It can be obtained as a nucleic acid fragment by performing PCR amplification using a nucleic acid as a template. The gene encoding wheat glutenin GluD1 y type No. 10 of the present invention is a gene encoding a wheat glutenin GluD1 y type No. 10 using a nucleic acid derived from wheat (preferably cultivar Max) exhibiting high bread-making properties as a template. By performing hybridization using a partial DNA fragment as a probe, it can be obtained as a nucleic acid fragment. The nucleic acid used as a template in these methods may be genomic DNA extracted by a conventional method from, for example, wheat varieties exhibiting high bread-making properties, such as Max, Cheyenne, or reverse transcription from mRNA extracted by a conventional method. It may be a synthesized cDNA or the like. The nucleic acid used as a template may be purified genomic DNA, purified cDNA, cDNA library, genomic DNA library, or the like. Alternatively, the gene encoding wheat glutenin GluD1 y type 10 of the present invention may be synthesized as a nucleic acid fragment by various nucleic acid sequence synthesis methods known in the art such as chemical synthesis methods.

  Furthermore, a desired mutation may be introduced into the base sequence (and thus the encoded amino acid sequence) of the gene encoding wheat glutenin GluD1 y type 10 obtained by site-directed mutagenesis. In order to introduce a mutation into a gene, a known method such as the Kunkel method or the Gapped duplex method, or a method equivalent thereto can be employed. For example, using a mutagenesis kit (for example, Mutan-K (TAKARA) or Mutan-G (TAKARA)) using site-directed mutagenesis, or TAKARA LA PCR in vitro Mutagenesis Mutation is introduced using a series kit.

  Preparation of mRNA used in the present invention, cDNA preparation, PCR, RT-PCR, library preparation, ligation into vector, cell transformation, determination of DNA base sequence, nucleic acid chemical synthesis, N-terminal of protein Experiments such as determination of the amino acid sequence on the side, mutagenesis, protein extraction, etc. can be carried out by the methods described in ordinary experiments. Examples of such experiments include Sambrook et al., Molecular Cloning, A laboratory manual, 2001, Eds., Sambrook, J. & Russell, DW. Cold Spring Harbor Laboratory Press.

  In the present invention, the term “high bread-making property” is used for plants, plant tissues, cells, flour, glutenin, which is a component of flour, and the like. In the present invention, “high bread-making property” means that when bread is produced using flour obtained from the plant or plant tissue as a raw material, the flour, or flour containing the glutenin in a high ratio, the comparison with the control sample This means that the bread volume increases. For example, when a bread is produced by using a wheat flour by a medium seed dough method using dry yeast as baker's yeast, a commercially available strong flour (for example, a high-grade bread flour from a Japanese flour company) is used instead. When the bread volume is 100% and the bread volume is increased to 108% or more, preferably 110 to 200%, the flour or the plant as the raw material of the flour is “high bread-making”. In the determination of “high bread-making property”, it is preferable to confirm improvement in sensory evaluation in addition to an increase in bread volume.

2. Host Plant In the present invention, rice (Oryza sativa) is preferably used as a host plant into which a gene encoding wheat glutenin GluD1 y type 10 is introduced. Rice varieties are not particularly limited, but examples of preferred rice varieties include “Nihonbare”, “Hoshinoyume”, “Kirara 397”, “Yukihikari”, “Yuki "Yukimaru", "Honoka224", "Dontokoi", "Koshihikari", "Sasanishiki", "Hitomebore", "Akitakomachi", `` Haenuki '', `` Kinuhikari '', `` Mutsuhomare '', `` Hinohikari '', `` Tsugaruotome '', `` Tsugaruroman '', `` Yume Akari ( `` Yumekakari '', `` Hanaechizen '', `` Yumetsukushi '', `` Hatsushimo '', `` Domannaka '', `` Kakehashi '', `` Iwatekko '', `` Iwatekko '' `` Manamusume '', `` Me Koina (Menkoina) "," Chiyonishiki (Chiyonishiki) "," Fukumirai (Fukumirai) ", and the like. These rice varieties can be obtained from, for example, Genebank, National Institute for Agrobiological Sciences.

3. Recombinant vector containing a gene encoding wheat glutenin GluD1 y type 10 The gene of the present invention obtained as described in 1 above can be inserted into a vector to produce a recombinant vector.

  The recombinant vector of the present invention can be prepared by ligating (inserting) a gene encoding wheat glutenin GluD1 y type 10 into an appropriate vector. To insert a gene encoding wheat glutenin GluD1 y type 10 into a vector, first cut the purified DNA with an appropriate restriction enzyme and insert it into the restriction enzyme site or multiple cloning site of an appropriate vector DNA. A method of ligation using ligase is employed. The vector into which the gene is inserted is not particularly limited and includes, for example, plasmid DNA, phage DNA, etc., but is preferably a vector for plant transformation such as plasmid DNA derived from Rhizobium, binary vector. In the present invention, the recombinant vector containing a gene encoding wheat glutenin GluD1 y type 10 is more preferably a vector that can be integrated into the genome of the introduced rice cell. Such a recombinant vector of the present invention introduces a gene that encodes wheat glutenin GluD1 y type 10 into rice and expresses wheat glutenin GluD1 y type 10 in rice cells, thereby allowing rice to produce a high Sex can be imparted. Therefore, the present invention also relates to a recombinant vector containing a gene encoding wheat glutenin GluD1 y type 10 for imparting high bread-making properties to rice.

  The recombinant vector of the present invention containing a gene encoding wheat glutenin GluD1 y type 10 includes, in addition to a sequence encoding wheat glutenin GluD1 y type 10, a constitutive or transient sequence for expressing the gene. Sex promoter sequences (such as the 35S promoter from cauliflower mosaic virus and promoters of heat shock-inducing proteins), selectable marker genes that facilitate the selection of transformants, and the origin of replication for using the Rhizobium binary vector system (Ti Or a Ri plasmid-derived replication origin). Examples of selection markers include drug resistance genes such as hygromycin resistance gene, dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene, kanamycin resistance gene and the like.

4). Production of transformed rice by introduction of a gene encoding wheat glutenin GluD1 y type 10 To produce the transformed rice of the present invention, a gene encoding wheat glutenin GluD1 y type 10 is introduced into rice cells, What is necessary is just to acquire the transformed rice cell. Moreover, the plant body of transformed rice can be produced by regenerating the transformed rice cells.

  In the present invention, “gene introduction” means that the gene is introduced into a cell of the host plant (ie, rice) in a form that can be expressed by, for example, a known genetic engineering technique. The gene introduced here may be incorporated into the genomic DNA of the host plant or may be present as it is contained in the foreign vector.

  A method for introducing a gene encoding wheat glutenin GluD1 y type 10 into plant cells is not limited. For example, the Agrobacterium method, the particle gun method, the electroporation method, the polyethylene glycol (PEG) method Plant transformation methods that are already established and widely used in the technical field of the present invention, such as microinjection method and protoplast fusion method, can be used. These plant transformation methods can be carried out according to the descriptions in general textbooks such as “Isao Shimamoto, Kiyotaka Okada“ Experimental Protocol for Model Plants From Genetic Methods to Genome Analysis ”(2001) Shujunsha). it can. For example, when the Agrobacterium method is used, an Agrobacterium-derived plasmid vector (for example, pBI101 or pBI121) or a binary vector (pPZP2H) into which a gene encoding wheat glutenin GluD1 y type No. 10 prepared as described in 3 above is incorporated. -lac) can be introduced into a Rhizobium, such as Rhizobium radiobactor, and the soil bacteria can be inoculated into and infected with rice calli to obtain rice transformants. Several variations of the Agrobacterium method have been developed in recent years (for example, Hiei Y, et al., Plant J., 6: 271 (1994); Toki et al. Disclosed in International Publication WO 01/06844). As an example, for example, ripened rice seeds to be transformed are sterilized, callus is induced from the seeds, cultured, and co-cultured with Rhizobium radiobacter introduced with a binary vector to infect them. By the method, a gene encoding wheat glutenin GluD1 y type 10 can be introduced into rice cells. Subsequently, Rhizobium adhering to the callus is washed, the callus is cultured in a selective medium, and the surviving callus is transformed into a regeneration medium (appropriate concentration of plant hormone (auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc. ), The plant body can be selectively regenerated from the transformed rice cells.

  When using the polyethylene glycol method, first remove the cell wall by lysing it with an enzyme, make it a protoplast, introduce a gene encoding wheat glutenin GluD1 y type No. 10 into the cell using polyethylene glycol, It may be regenerated to the body (Datta SK: In Gene Transfer To Plants (Potrykus I and Spangenberg, Eds) pp. 66-74 (1995)). In this method, Indian rice varieties are more suitable for introducing the gene.

  When using the electroporation method, the cell wall is first removed by lysing with an enzyme to make a protoplast, and then a gene encoding wheat glutenin GluD1 y type 10 is introduced into the cell by applying an electric pulse, and then What is necessary is just to reproduce | regenerate to a plant body (Toki S, et al., Plant Physiol., 100: 1503 (1992)). In this method, Japanese rice varieties are more suitable for introducing the gene. In the electroporation method, the gene encoding wheat glutenin GluD1 y type 10 may be in the form of the gene itself or may be incorporated into a vector.

  In the case of using the particle gun method, the rice plant body, the rice organ, and the rice tissue itself may be used as they are, may be used after preparing a section, or may be used after preparing a protoplast (Christou). P, et al., Biotechnology 9: 957 (1991)). Using a gene transfer device (eg PDS-1000 (BIO-RAD), etc.) for the sample prepared in this way, fine particles such as gold or tungsten coated with a gene encoding wheat glutenin GluD1 y type 10 ( The gene is introduced into the rice cells by spraying with a high pressure gas with a diameter of about 1 to 2 μm). Treatment conditions vary depending on the plant or sample, but are usually performed at a pressure of about 450 to 2000 psi and a distance of about 4 to 12 cm. In the particle gun method, the gene encoding wheat glutenin GluD1 y type 10 may be in the form of the gene itself or may be incorporated into a vector.

  Confirmation of whether or not the gene encoding wheat glutenin GluD1 y type 10 has been incorporated into rice cells can be performed by well-known methods such as PCR, Southern hybridization, Northern hybridization. For example, genomic DNA is prepared from transformed rice and PCR is performed using a primer that can specifically amplify a gene encoding wheat glutenin GluD1 y type # 10. The amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR Green solution, etc., and the amplified product is detected as a band of the expected size. It can be confirmed that a gene encoding wheat glutenin GluD1 y type 10 was introduced into rice cells. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. In addition, a method in which an amplification product is bound to a solid phase such as a microplate and the amplification product is confirmed by fluorescence or an enzyme reaction can be employed. Furthermore, protein is extracted from the rice cells, fractionated by two-dimensional electrophoresis, and wheat glutenin GluD1 introduced into rice cells is detected by detecting a protein band considered to be wheat glutenin GluD1 y type # 10. It is suggested that y type 10 is expressed, that is, the rice cells are transformed. Subsequently, the N-terminal amino acid sequence of the detected protein is determined by Edman degradation or the like, and whether or not it matches the N-terminal sequence of SEQ ID NO: 2 is further verified for the transformation of the rice cell. be able to.

  In the present invention, the “transformed rice” may be a transformed cell of rice having a gene encoding the introduced wheat glutenin GluD1 y type 10 in the cell in a state where it is expressed or can be expressed. . Alternatively, the transformed rice of the present invention is the whole plant or organ (eg, leaf, petal, stem, root, cereal (seed), etc.), tissue (eg, epidermis) of rice that contains such transformed cells at least in part. Phloem, soft tissue, xylem, vascular bundle, fence-like tissue, spongy tissue, etc.) or cultured cells (eg, callus). The transformed rice of the present invention is preferably one in which wheat glutenin GluD1 y type No. 10 is accumulated in the grain (seed). The term “transformed rice” includes not only transformed cells into which a gene has been introduced, but also callus in which the gene has been grown, plants regenerated from the callus, and parts of regenerated plants. The transformed rice plant of the present invention includes a progeny plant obtained by sexual reproduction or asexual reproduction of a plant into which the gene is introduced (including a plant regenerated from a transformed cell or a callus), And a part of the tissue or organ of the progeny plant body (seed, protoplast, etc.). The transformed rice of the present invention obtains a propagation material such as grain (seed) and protoplast from a rice plant transformed by introducing a gene encoding wheat glutenin GluD1 y type 10 and cultivates or It can be mass-produced by culturing.

  Wheat glutenin GluD1 y type No. 10 that accumulates in the grain of the transformed rice of the present invention is a glutenin subunit that imparts high bread-making properties. Therefore, the grain of the transformed rice of the present invention can be used as a raw material for a high-quality flour substitute. That is, the grain of the transformed rice of the present invention can be used as a grain for breadmaking.

  The results of the present invention as described above provide a means for domestic production of flour substitute plants (bread rice) that depend on imports, using paddy fields in Japan that are subject to cropping restrictions. It provides a stable supply of bread crumbs.

EXAMPLES Hereinafter, although this invention is demonstrated concretely using an Example, the technical scope of this invention is not limited to these Examples.
(1) Isolation of a gene encoding wheat glutenin GluD1y type 10 Genomic DNA was extracted from wheat cultivar Max (Triticum aestivum; distributed by Hokuren Agricultural Cooperative Federation) showing high bread-making properties according to a conventional method. Next, using this as a template, a gene (3275 base pairs) encoding wheat glutenin GluD1 y type 10 was amplified by PCR. The primer sequences used for this PCR were as follows.

Forward primer GluD1-2b-2321U: CGC GTC ACA GAA CAG ATA (SEQ ID NO: 3)
Reverse primer GluD1-2b-5579L: TAT GCG TTA TTC AGT AAG (SEQ ID NO: 4)
The PCR reaction conditions were as follows: 94 ° C. for 1 minute → 61 ° C. for 1 minute → 72 ° C. for 2 minutes (30 cycles).

  The amplified fragment of 3275 base pairs thus obtained was subcloned into a pCR2.1-TOPO vector (Invitrogen Corporation) by a conventional method, and the resulting clone was named TOPO-GluDly10. Next, the base sequence of the entire amplified fragment in TOPO-GluDly10 is determined, and the amplified fragment is a gene encoding wheat gluten GluD1 y type No. 10 whose base sequence has already been reported (Non-patent Document 1, Accession No. X12929). ) Was confirmed. The base sequence of the gene encoding the isolated wheat gluten GluD1 y type 10 is shown in SEQ ID NO: 1.

(2) Preparation of transgenic rice introduced with a gene encoding wheat glutenin GluD1y type 10 Cut clone TOPO-GluDly10 with EcoR1, cut out a genomic fragment containing the gene encoding GluD1 y type 10 It was inserted into the SmaI site of pPZP2H-lac (Plant Biotechnology 18, 219-222, 2001), a binary vector for plant transformation. The binary vector thus obtained was named GluD1yMfull. This binary vector was introduced into Rhizobium radiobacter EHE101 (distributed from Riken Genebank) and then transformed into rice cultivar Nipponbare (Oryza sativa) according to the Agrobacterium-mediated transformation method (method of Toki et al .: International Publication WO 01/06844). ; Infected with the callus of the National Institute of Agrobiological Sciences, Genebank Genetic Resources Management Division, and selected regenerated individuals in the presence of hygromycin.

(3) Confirmation of wheat glutenin GluD1 y type 10 protein accumulated in the grain of transformed rice Grain was collected from the transformed rice plant body, and the total protein was extracted from the endosperm portion by a conventional method. The extracted protein is subjected to SDS-PAGE electrophoresis using a 10-20% gradient polyacrylamide gel, and all proteins are stained with the staining reagent CBB-R250. This was confirmed (arrow in FIG. 1).

  In FIG. 1, (A) is an overall view of an electrophoretic photograph. The protein bands predicted to be wheat glutenin are indicated by arrows. (B) is an enlarged view of an electrophoretic photograph (A) in the vicinity where wheat glutenin protein is accumulated. * Indicates a band presumed to be wheat glutenin. In both FIGS. (A) and (B), the sample in the leftmost lane (NB) is the total protein (control sample) extracted from non-transformed Nipponbare seeds. The samples in the 1st to 6th lanes indicated by the lines in the upper part of the photograph are all proteins extracted from seeds that are cultivated in the plant of transformed rice into which a gene encoding wheat glutenin GluD1 y type 10 has been introduced. As can be seen from these results, a band presumed to be wheat glutenin was detected in transgenic rice introduced with a gene encoding wheat glutenin GluD1 y type # 10. On the other hand, the band was not detected in non-transformed rice.

  In order to confirm that the protein of the band presumed to be wheat glutenin is a translation product derived from the gene that was truly introduced, this fraction was extracted from the gel and subjected to two-dimensional electrophoresis to obtain a higher accuracy. I drew it. FIG. 2 shows the result of eluting the protein of the band presumed to be wheat glutenin (GluD1 y type No. 10) shown in FIG. 1 and further fractionating it by two-dimensional electrophoresis. The protein presumed to be wheat glutenin (GluD1 y type No. 10) detected only in the rice-derived sample into which the gene was introduced is indicated by an arrow. Thus, the result of two-dimensional electrophoresis also showed the presence of a protein that can be detected only in rice into which a gene encoding wheat glutenin GluD1 y type 10 was introduced.

Furthermore, a protein presumed to be wheat glutenin fractionated by two-dimensional electrophoresis was extracted from the gel, and the N-terminal amino acid sequence was analyzed by Edman degradation. As a result, the N-terminal sequence of this protein is
Trp-Gln-Glu-Pro-Gly-Gln-Gly-Gln-Gln-X-Tyr-Tyr (SEQ ID NO: 5)
It was shown that. In this sequence, X refers to an amino acid that could not be identified. This sequence coincided with the N-terminal of the amino acid sequence (SEQ ID NO: 2) of the transgenic wheat glutenin (GluD1 y type No. 10).

  From the above results, it was confirmed that the protein specifically expressed in the transformed rice and accumulated in the grain was the gene product of the gene encoding the introduced wheat glutenin GluD1 y type No. 10.

  The transformed rice containing the gene encoding wheat glutenin GluD1 y type No. 10 of the present invention is a plant capable of producing rice grains accumulating wheat glutenin protein in high yield even under Japanese climatic conditions. It can be used advantageously. By using the transformed rice of the present invention, it is possible to harvest cereal (bread rice) that can be used as a substitute for wheat flour in paddy fields where production of rice is restricted by reduction. That is, the present invention not only provides new rice varieties, but also provides alternative plants for wheat crop failures caused by continuous cropping failures that are fatal to the field farming method. Therefore, the present invention not only contributes to domestic agricultural promotion, but also enables the supply of new agricultural resources to industries that use wheat from an international perspective.

It is a photograph showing the result of subjecting the total protein extracted from rice seeds to SDS-PAGE electrophoresis and staining the fractionated total protein. FIG. 2 is a photograph showing the results of elution of a protein in a band presumed to be wheat glutenin (GluD1 y type No. 10) shown in FIG. 1 and further fractionating it by two-dimensional electrophoresis.

  The sequences of SEQ ID NOs: 3 and 4 are primers.

Claims (4)

A transformed rice comprising the following gene (a) or (b) encoding wheat glutenin protein GluD1 y type 10:
(a) a gene consisting of DNA having the base sequence represented by SEQ ID NO: 1, or
(b) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2   The transformed rice according to claim 1, wherein the rice variety is Nipponbare. A method for producing a high bread-producing transformed rice characterized by introducing the following gene (a) or (b) encoding wheat glutenin protein GluD1 y type 10 into rice cells.
(a) a gene consisting of DNA having the base sequence represented by SEQ ID NO: 1, or
(b) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 A recombinant vector for imparting high bread-making property to rice, comprising the following gene (a) or (b) encoding wheat glutenin protein GluD1 y type 10:
(a) a gene consisting of DNA having the base sequence represented by SEQ ID NO: 1, or
(b) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2

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